1. Field of the Invention
The present invention relates to DNA molecules which code for fusion proteins from enzymatically active fractions and polypeptide fractions which can be cleaved therefrom, expression systems which contain these DNA molecules and the use thereof as test systems for inhibitors of viral proteases.
2. Description of the Background Art
Rhinoviruses are ss(+)RNA viruses and represent a genus within the Picornaviridae (Cooper, P.D. et al., Intervirology 1:165-180; (1978); MacNaughton, M. R., Current Top. Microbiol. Immunol. 97:1-26 (1982)). They are widespread, attack the upper respiratory tract in humans and result in acute infections which lead to colds, coughs, sore throat, etc. and are generally referred to as colds (Stott, E. J. et al., Ann Rev. Microbiol. 26:503-524 (1972)). Infections caused by rhinoviruses are among the commonest diseases in man. Admittedly, the illness is usually harmless but because of the temporary weakening of the body, secondary infections caused by other viruses or bacteria occur, which may under certain circumstances, result in serious illness. Of the total of about 115 different know serotypes of human rhinoviruses, until now, 3 serotypes have been closed and completely sequenced: German Patent Application P 35 05 148.5; Skern; T. et al., Nucleic Acids Res. 13:2111-2126 (1985); Duchler, M. et al., Proc. Natl. Acad. Sci. U.S.A. 84:2605-2609 (1987); Stanway, G. et al., Nucleic Acids Res. 12:7859-7877 (1984); Callahan, P. L. et al., Proc. Natl. Acad. Sci. U.S.A. 82:732-736 (1985)).
Comparison of the amino acid sequences of the individual proteins shows that the viral enzymes are particularly well preserved. Thus, the homology between the protease P2A of HRV89 and HRV2 is about 85%; in protease P3C, 75% of the amino acids are identical (Duchler, M. et al., (1987)). These levels are substantially above the average percentages observed in the protein as a whole. It can, therefore, be assumed that it is precisely the viral enzymes which are particularly well preserved in evolution and are very similar in their properties in different rhinoviruses.
Hardly any other viral system is so dependent in its regulation of the course of infection, on a controlled limited proteolysis as that of the picornaviridae. The genomic single-stranded (+)RNA of the rhinoviruses is modified shortly after infection by the cleaving of the oligopeptides VPg bound to the 5' end which serves as mRNA for the synthesis of a polyprotein which includes the entire continuous reading frame of the nucleic acid sequence (Butterworth, B. E., Virology 56:439-453 (1973); McLean, C. et al., J. Virol. 11:341-344 (1973); McLean, C. et al., J. Virol, 19:903-914 (1976)). The mature viral proteins are formed exclusively by proteolytic cleaving from this polyprotein, the effective proteases themselves being part of this polyprotein. The first step in this processing is the cleaving of the precursor stage of the coat proteins which is effected by the protease P2A. In the succession of the genes, the sequence of the protease P2A is immediately after the fragment coding for the coat protein. P2A is, therefore, the first detectable enzymatic function of the virus because of its location in the polyprotein.
To some extent, P2A cleaves automatically from the precursor of the coat proteins and is responsible for the separation of the capsid precursor P1 from the rest of the polyprotein. Separation of the coat protein region from the fragment responsible for replication is already taking place during translation of the polyprotein.
This step is essential for the further progress of the viral infection. It is known of the polio virus system that, in all probability, all the enzymes involved in this maturation cleaving are virally coded (Toyoda, H. et al., Cell 45:761-770 (1986)). In the polio virus, there are three types of cleaving signals (FIG. 1); the Q-G site which is used most and which is recognized by the viral protease P3C, and the Y-G site which is used by P2A as a recognition signal. Initially, the protease P3C was of central interest in explaining the proteolytic processing of picorna viruses. Very early on, it was possible to describe a proteolytic activity equivalent to P3C in EMC (Pelham, H. R. B., J. Biochem. 85:457-461 (1978); Palmenberg, A. C. et al., J. Virol, 32:770-778 (1979)). In the course of further investigations, it was found that the leader peptide (L) of cardio viruses (e.g., EMCV) and aphto viruses (e.g., FMDV) which is not present in rhino- and entero viruses, is involved in proteolytic processing of EMCV (Palmenberg, A. C., J. Cell, Biochem. 331191-1198 (1987)). It was subsequently possible to demonstrate, by isolating polio P3C and using immunological methods, that P3C autocatalytically cuts itself out of the polyprotein in order to attack all potential Q-G cleaving sites in "trans".
The use of recombinant systems which represented, inter alia, the P3C region, made it possible to express the P3C of some entero- and rhino viruses (Werner, G. et al., J. Virol, 57:1084-1093 (1986)) and accurately to characterize the P3C of polio (Hanecak, R. et al., Cell 37:1037-1073 (1984); Korant, B. D. et al., Biomed. Biochim. Acta 45:1529-1535 (1989)) and the equivalent proteolytic function in FMDV (Klump, W. et al, Proc. Natl, Acad. Sci. U.S.A. 81:3351-3355 (1984); Burroughs, J. N. et al., J. Virol. 50:878-883 (1984)). By mutagenesis studies in vitro, it was possible to demonstrate that the replacement of the highly preserved amino acids cysteine (position number 147) and histidine (position number 161) in P3C of polio virus leads to an inactive enzyme, whereas the mutation of htenon-conversed cysteine (position number 153) has no appreciable effect on the proteolytic activity of polio P3C. It was further concluded that polio P3C belongs to the cysteine proteases (Ivanoff, L. A. et al., Proc. Natl. Acad. Sci. U.S.A. 83:5392-5396 (1986)). It was also possible to show, by in vitro mutagenesis of polio P3C (i.e., by replacement of the preserved valine by alanine in position 54 of the protease) that this mutation in a full size cDNA of polio after transfection into COS1 cells results in a polymerase-deficient virus (Dewalt, P. G. et al., J. Virol. 61:2162-2170 (1987)).
Antibodies developed against poliko P3C did admittedly prevent any cleaving carried out at Q-G, but did not prevent cleaving between Y-G (Hanecak, R. et al., Proc. Natl., Acad. Sci. U.S.A. 79:3973-3977 (1982)). This observation lead to the conclusion that proteolytic processing at Y-G sites requires its own protease. The seat of this second proteolytic activity was clearly identified as being in P2A within the polio virus. It was interesting to discover that P2A carries out alternative cleaving in the protease-polymerase region (3CD) which also takes place at a Y-G site. However, this cleaving would appear not to have any biological significance curing replication of the virus (Toyoda, H. et al. loc. cit.). Since the synthesis of the host protein is very rapidly stopped during infection with polio virus in Hela cells, but the translation of the polio virus RNA can proceed unimpeded, it was assumed that one or more regulating factors of the translation were altered during the infection. In fact, earlier findings show that the eukaryotic initiation factor 4F is changed by proteolytic cleaving of the p220 component during polio virus infection in Hela cells (Etchison, D. et al., J. Virol. 51:832-837 (1984); Etchison, D. et al., J. Biol. Chem. 257:14806-14810 (1982)). Subsequently, it was shown that P2A is indirectly responsible for this modification of p220 in infected cells (Krausslich, H. G. et al., J. Virol. 61:2711-2718 (1987). The question as to the transactivity of the two proteases P3C and P2A could thus be answered in the affirmitive in the polio virus system insofar as polio viruspolypeptide precursors expressed in vitro which contained the proteolytic recognition sequences were able to be processed by exogenic P3C or P2A proteases (Nicklin, M. J. H. et al., Proc. Natl. Acad. Sci. U.S.A. 84:4002-4006 (1987)).
It was also very interesting to discover that two proteins similar to the picornaviral proteases P3C and P2A were discovered in the plant viral system of Comoviridae (Cowpea Mosaic Virus) (Garcia, J. A. et al., Virology 159:67-75 (1987); Verver, J. et al., EMBO 6:549-554 (1987)). These two viral proteins are involved in the proteolytic processing of the two polyproteins coded by two separately packed ss (+)RNA molecules (B and M RNA), the two Cowpea mosaic virus proteases showing great similarity to the picorna viruses in sequence and cleaving specificity. This remarkable homology of non-structural proteins between Picorna and Comoviruses not only indicates a genetic relationship between these two families of virus but also shows how essential viral proteolytic processing is for these two families of viruses.
The third type of viral maturation cleaving, namely that of VPO (precursor protein of VP2 and VP4), has been described, in the case of Mengo and Rhino virus, with the aid of X-ray structural data. This latter proteolytic event in viral maturation appears to be based on an unusual autocatalytic serine protease type in which basic groups of the viral RNA participate in the formation of the catalytic center, these basic groups acting as proton acceptors (Arnold, E. et al., Proc. Natl., Acad. Sci. U.S.A. 84:21-25 (1987)).
The cleavage site specificity of the viral proteases was determined in the polio virus system by N-terminal sequencing of the majority of polio virus proteins (Pallansch, M. A. et al., J. Virol. 49:873-880 (1984)). By cloning and sequencing HRV2 (Skern, T. et al., Nucleic Acids Res. 13:2111-2126 (1985), it was possible to derive the majority of cleavage sites by sequence comparisons with polio virus and HRV14. Furthermore, the position of the cutting sites between VP4/VP2, VP2/VP3 and BP3/VP1 could be determined by N-terminal sequencing of VP2, VP3 and VP1. The cleavage signal between VP1 and P2A was partly determined by C-terminal sequencing of VP1 (Kowalski, H. et al., J. Gen. Virol. 86:3197-3200 (1987). Thus, five different cleavage signals were found in HRV2: Q-S, Q-G, Q-N, A-G and E-S (FIG. 2).
Cysteine proteases are widespread in nature (e.g., papain, cathepsin B, H and S), and their characterization and inhibition is of great scientific and therapeutic value (for a survey see Turk, V., 1986, Cysteine Proteinases and their Inhibitors, Walter de Gruyter; Barrett, A. J. and Salvesen, G., 1986, Proteinase Inhibitors, Elsevier). In the pivorna viral system, too, all kinds of inorganic and organic compounds as well as peptide derivatives and proteins are now known which have an inhibitor effect on the proteolytic processing of these viruses. The effect of these substances is based on the direct interaction with the proteases (Kettner, C. A. et al., U.S. Pat. No.: 4,652,552 (1987); Korant, B. D. et al., J. Cell. Biochem. 32:91-95 (1986)) and/or on the indirect route of interaction with substrates of these proteases (Geist, F. C. et al., Antimicrob. Agents Chemother, 31:622-624 (1987); Perrin, D. D. et al, Viral Chemotherapy 1:288-189 (1984)). The problem with the majority of these substances is the relatively high concentration needed for inhibition and the in some cases high toxicity of these compounds.